We recently described a family of NADP H-oxidases, the Nox enzymes that have been implicated in cancer, particularly colon and prostate cancers. The enzymes are flavocytochromes that produce reactive oxygen species (ROS) and one of these, hydrogen peroxide, has functions as a signal molecule, stimulating mitosis and angiogenesis. Aberrant epithelial expression or activation of these enzymes has therefore been proposed to play a role in cancer progression. However, to date, little is known about how the activity of these enzymes is regulated. This application therefore focuses on the molecular mechanisms of regulation of Nox1, Nox3, Nox4 and Nox5. The Nox enzymes are homologs of gp91phox, the catalytic subunit of the phagocyte respiratory burst oxidase. The latter is regulated by catalytic subunits p47phox and p67phox, and we recently identified novel homologs of these subunits termed NOXO1 (Nox Organizing Protein 1) and NOXA1 (Nox Activating Protein 1) from colon and testis cDNA libraries. Evidence points to these proteins as regulatory subunits of the Noxl and possibly Nox5. We have identified 4 isoforms (splice forms) of NOXO1 and 5 splice forms of NOXAI. In addition, NOXO1 and NOXA1 contain Src-homology 3 (SH3) domains and proline-rich regions, and NOXA1 contains a 4 tandem copies of a TPR motif that is proposed to participate in heterodimer formation with Rac or another small GTP-binding protein. Thus, the role of these subunits and their interaction domains in the regulated assembly/activation of Noxl-5, and the role of small GTPases will be investigated. Nox5 contains a domain similar to the recoverin group of calcium-binding proteins, and is presumed to be regulated by calcium. In addition, Nox5 contains a proline-rich motif that is predicted to interact with an SH3 domain like the one in NOXO1. The role of regulatory subunits and calcium as alternative or synergistic mechanisms for regulating Nox5 will be investigated. These studies are expected to have implications with regard to both normal and aberrant generation of ROS signals relevant to cell growth and cancer.